Summary

During eye development, retinal progenitors are drawn from a multipotent,
proliferative cell population. In Drosophila the maintenance of this
cell population requires the function of the TALE-homeodomain transcription
factor Hth, although its mechanisms of action are still unknown. Here we
investigate whether members of the Meis gene family, the vertebrate homologs
of hth, are also involved in early stages of eye development in the
zebrafish. We show that meis1 is initially expressed throughout the
eye primordium. Later, meis1 becomes repressed as neurogenesis is
initiated, and its expression is confined to the ciliary margin, where the
retinal stem population resides. Knocking down meis1 function through
morpholino injection causes a delay in the G1-to-S phase transition of the eye
cells, and results in severely reduced eyes. This role in cell cycle control
is mediated by meis1 regulating cyclin D1 and c-myc
transcription. The forced maintenance of meis1 expression in cell
clones is incompatible with the normal differentiation of the
meis1-expressing cells, which in turn tend to reside in
undifferentiated regions of the retinal neuroepithelium, such as the ciliary
margin. Together, these results implicate meis1 as a positive cell
cycle regulator in early retinal cells, and provide evidence of an
evolutionary conserved function for Hth/Meis genes in the maintenance of the
proliferative, multipotent cell state during early eye development.

Recent work points to a role for Meis genes in eye development: Meis1 and
Meis2 are upstream regulators of Pax6 in the developing lens in chicken and
mouse (Zhang et al., 2002),
and mouse embryos homozygous for a homeodomain-less Meis1 gene show
eye malformations (Hisa et al.,
2004). Still, the precise role(s) played by Meis genes during eye
development remain(s) unknown. If the parallels in early eye development
between flies and vertebrates hold true for Hth/Meis, Meis genes might be
involved in stimulating proliferation, or preventing premature differentiation
in the optic primordium, or both. Here, we investigated these hypotheses in
the zebrafish (Danio rerio).

MATERIALS AND METHODS

Probe preparation, in situ hybridization and immunolabeling

Antisense RNA probes were prepared from cDNAs and labeled with digoxigenin.
Specimens were fixed, hybridized and stained as described
(Tena et al., 2007).

In vitro RNA synthesis and microinjection of mRNA and
morpholinos

cDNAs were linearized and transcribed as described
(Tena et al., 2007). One- to
two-cell-stage zebrafish embryos were injected in the yolk with mRNA and/or
morpholino (MO) diluted in ∼5 nl of injection solution (10% Phenol Red in
DEPC-treated water).

MOs targeting the ATG region of meis1, meis2.2, meis3 and
meis4 mRNAs (see Fig. S1A in the supplementary material) were
synthesized by GeneTools. We verified the target specificity of
meis1- and meis2.2-MOs in Xenopus laevis assays
(see Fig. S1B in the supplementary material), and the biological specificity
of the meis1-MO by testing its ability to reduce the rhombomere-3
expression of krox20 (also known as egr2 - ZFIN) (see Figs
S2 and S5 in the supplementary material).

As controls, we injected similar amounts (8-16 ng) of a control MO directed
against the Xenopus tropicalis olig2 gene that shows no match in the
zebrafish genome (see Fig. S1 in the supplementary material). The
meis3-MO, which has nine and seven mismatches with meis1 and
meis2.2, respectively, also served as control MO in some
experiments.

meis1 retracts accompanying the ath5 wave and becomes
restricted to the CMZ. (A-D) meis1 and (E-H)
ath5 expression analyzed by single in situ hybridization.
Developmental stages are indicated as hours post-fertilization (hpf) at
28.5°C. Lateral views of whole-mount (A,E) or dissected (B-D,F-H) eyes,
with dorsal up and anterior to the left. The front of the ath5 domain
is marked by black arrowheads. The red arrowhead in D points to meis1
expression in the prospective ciliary margin. (I-M) Transverse 40 μm
vibratome sections. (I,J) Dorsal is up. meis1 is weakly expressed in
the lens ectoderm before its thickening (I), but no signal is detected once
the lens placode is formed (J). (K-M) Dorsal is to the left. meis1
and ath5 expression domains are complementary as shown by double in
situ hybridization (K,L). Approximate limits of the ath5 signal are
indicated by the black arrowheads. (M) At 42hpf, meis1 expression is
detected by in situ hybridization in the ciliary margin (red arrowheads) and
in the postmitotic ganglion cells (black arrowhead). L, lens.

Eye phenotype measurements

The polygonal-lasso tool from Adobe Photoshop was used to measure in
digital photographs taken with the same magnification, the eye surface area
(in pixels) of control and morphant embryos. The volume of each eye was
estimated considering it as a hemisphere of radius equal to the radius of a
circle with that same area. Measurements from 20 eyes for each condition were
compared using a χ2 test.

The PCR fragments were subcloned into pGEMT-Easy (Promega) and sequenced.
Meis cDNAs were cloned into pCS2 MT, pCS2p+MTC2 or pCS2eGFP (kindly provided
by D. Turner, University of Michigan, USA) to generate N-terminal (Myc-meis)
and C-terminal (meis-Myc) Myc-tagged meis or N-terminal GFP-tagged
meis1 (GFP-meis1), respectively. To generate the
Tol2-GFP-meis1 and Tol2-GFP constructs, we inserted the
GFP-meis1 and GFP fragments, respectively, into SalI and
SspI sites of Tol2 (pT2KXIG).

Acridine Orange staining

DNA content analysis and flow cytometry

Eyes dissected from 19hpf zebrafish embryos were disaggregated, and PI
staining carried out as described
(Langenau et al., 2003). DNA
content was analyzed on a BD FACSAria and results processed with FloJo
software (Tree Star). A χ2 test was used for statistical data
analysis.

Induction of ectopic expression mosaics

The Tol2 transposon/transposase method of transgenesis
(Kawakami et al., 2004) was
used with minor modifications. Four- to 16-cell-stage zebrafish embryos were
injected in the yolk with 5-12.5 pg of either Tol2-GFP-meis1 or
Tol2-GFP constructs, plus 125 pg of transposase-encoding mRNA in a final
volume of 5 nl of injection solution. Embryos were cultured at 28.5°C,
staged and fixed. Anti-GFP antibody was used to detect the GFP- or
GFP-meis1-expressing clones. A stack of confocal z-sections
was obtained for each eye analyzed. Three-dimensional reconstruction of the
stacks was used to determine the location of the clones.

RESULTS AND DISCUSSSION

meis1 expression is restricted to the undifferentiated and
proliferating cells of the early zebrafish eye

Of all five zebrafish Meis genes (meis1, 2.1, 2.2, 3 and
4.1), only meis1 and meis2.2 are expressed during
early stages of eye development (Kudoh et
al., 2001; Waskiewicz et al.,
2001; Zerucha and Prince,
2001; Thisse and Thisse,
2005) (this work). meis1, as monitored by in situ
hybridization, or by a YFP insertional reporter inserted close to
meis1, was seen to be uniformly transcribed in the eye primordium
from 15 to ∼24hpf (Fig. 1A
and see Fig. S3 in the supplementary material), a period in which all cells
proliferate (Li et al., 2000).
After this time, meis1 expression progressively retracted in the
retina (Fig. 1B-D,K,L) as the
neurogenic wave, marked by ath5 (also known as atoh7 - ZFIN)
expression, expands from antero-nasal to posterior-temporal positions
(Fig. 1F-H)
(Hu and Easter, 1999;
Li et al., 2000;
Masai et al., 2000).
meis1 remained transiently expressed in the ciliary margin zone
(CMZ), where the retinal stem population resides
(Fig. 1D,M). meis2.2
was also found to be expressed uniformly in early eye primordia, but its
expression faded away by 20hpf (see Fig. S3 in the supplementary material).
Similar to the situation found in chicken and mouse
(Zhang et al., 2002),
meis1 was expressed in the prospective lens ectoderm, but was turned
off as the lens placode started to thicken
(Fig. 1I,J). Therefore,
meis1 expression is associated with the undifferentiated,
proliferative cells during the early development of the zebrafish eye. In
addition, a new wave of Meis gene expression starts in postmitotic neurons at
around 36-42hpf (Fig. 1M and
see Fig. S3 in the supplementary material). Interestingly, at 4 days
post-fertilization (dpf), meis2.2 expression had replaced
meis1 at the CMZ.

meis1 is required for the growth of the eye primordium.
(A,B) Lateral views of representative 72hpf control-MO (A) and
meis1-MO (B) -injected fish. meis1 morphants are
microphthalmic. (C,D) Confocal images of dissected eyes stained
for propidium iodide (nuclei), rhodamine-phalloidin (filamentous actin) and
Islet1, which labels GCL nuclei and some in the INL. The reduced eyes from
meis1 morphants show apparently normal retina lamination
(D,D′), but fewer cells than control eyes (C,C′). Area (E)
and estimated volume (F) of control-MO and meis1-MO-injected
embryos at 72hpf. meis1- morphant embryos show a significant
(P<0.001) reduction in eye area and volume (45% and 60%,
respectively). n=20 for each condition.

meis1 is required to promote the G1-to-S transition of the
eye primordium cells, and regulates the transcription of cyclin D1
and c-myc

The early expression pattern of meis1 suggests that it plays a
role in the proliferative/multipotent cells of the developing eye. To
determine what role that is, we knocked down meis1 function using
meis1-specific morpholinos (meis1-MO). By the end of
embryogenesis, meis1 morphants were severely microphthalmic (>60%
of embryos injected with 8 ng of meis1-MO, n=163)
(Fig. 2A,B,E,F), with eyes
containing fewer cells than controls (Fig.
2C,D). Despite this, meis1-morphant eyes showed
apparently normal retinal lamination (Fig.
2C′,D′). The lens was normal or slightly reduced
(Fig. 2D and data not shown).
The co-expression of meis2.2 and meis1 during optic vesicle
stages suggested a possible functional redundancy between these two genes.
Nevertheless, injection of meis2.2-MO (8 ng/embryo) caused only mild
eye reductions in 22% of the treated embryos (n=299). Furthermore,
co-injection of equivalent amounts of meis1- and meis2.2-MOs
(4 ng of each MO/embryo) did not significantly enhance the penetrance or
severity of the microphthalmia (29%, n=196). These results suggest
that meis2.2 does not have a major role during early stages of eye
development in the zebrafish. The phenotype observed in meis1
morphants does not appear to be due to an abnormal eye primordium
specification. Although we found a slight decrease in pax6b
expression in meis1-morphant eyes, as estimated by RT-PCR (see Fig.
S4C,D in the supplementary material), the early expression of the eye selector
genes pax6b and rx2
(Stigloher et al., 2006)
seemed unaffected, when observed by in situ hybridization (see Fig. S4A,B in
the supplementary material and data not shown). In agreement with this, we
found that the expression of the Pax6 gene eyeless is independent of
hth during the development of the Drosophila eye (see Fig.
S4E,F in the supplementary material).

To further dissect the mechanisms underlying the observed microphthalmia,
we assessed whether meis1 controls the cell cycle.
meis1-morphant eyes, at 19hpf, had a significantly higher percentage
of cells in G1 phase than control embryos
(Fig. 3A,E), indicating a
requirement of meis1 in promoting the G1-S transition. Viability of
these cells was not compromised, as meis1-morphant eyes did not show
a significant increase in the levels of active Caspase 3, or in the vital
incorporation of Acridine Orange (not shown).

cyclin D1 (ccnd1) and c-myc (also known as
myca - ZFIN) are two major G1 regulators of the cell cycle in
vertebrates (Levine and Green,
2004). During the development of the zebrafish eye, cyclin
D1 and c-myc are first widely expressed in the optic vesicle,
followed by a progressive restriction to the proliferating cells of the neural
retina (Thisse and Thisse,
2005; Yamaguchi et al.,
2005), a pattern that is reminiscent of that of meis1. In
addition, recent work shows that cyclin D1 is required for
proliferation in the zebrafish developing retina, as cyclin D1
morphants are microphthalmic (Duffy et al.,
2005). The similarity between the patterns of expression of
meis1, cyclin D1 and c-myc, and the similar eye phenotypes
of cyclin D1 and meis1 morphants, prompted us to ask whether
cyclin D1 and c-myc were under meis1 control.
Indeed, meis1 morphants showed a dramatic reduction of cyclin
D1 and c-myc expression in the eye when compared with
control-injected embryos (Fig.
3F-I and see Fig. S5 in the supplementary material). In addition,
the co-injection of either cyclin D1 or c-myc mRNAs
partially rescued the cell cycle defects of meis1 morphants to levels
similar to those obtained by co-injection of GFP-meis1 mRNA
(Fig. 3B-E). These results
place cyclin D1 and c-myc functionally downstream of
meis1 in the control of cell cycle progression in the developing eye.
Whether meis1 regulates the transcription of cyclin D1 and
c-myc directly or indirectly is unknown.

Maintenance of meis1 expression is incompatible with cell
differentiation

In Drosophila, hth not only promotes proliferation in the eye
primordium, but forced maintenance of its expression results in a delay or
block of retinal differentiation (Pai et
al., 1998; Pichaud and
Casares, 2000; Bessa et al.,
2002). Similarly, in the early zebrafish eye, meis1
expression is found in undifferentiated cells but is turned off as
neurogenesis advances (Fig. 1).
To test whether maintaining meis1 expression is incompatible with
retinal differentiation, we analyzed the distribution of clones of cells
expressing either GFP or GFP-tagged-Meis1 in developing retinas, prior to and
after the initiation of neuronal differentiation
(Fig. 4 and see Fig. S6 in the
supplementary material). Differentiation was followed using the GCL marker
islet1. When analyzed between 24 and 30hpf, a stage at which most of
the retina is undifferentiated, all GFP- and 80% of GFP-Meis1-expressing
clones spanned the whole width of the neuroepithelium (n=57 and 46,
respectively; Fig. 4A,D). Later
in development, when retinal differentiation is ongoing and layering becomes
apparent, most GFP clones appeared in the central retina and contained both
Islet1-expressing and non-expressing cells (90%)
(Fig. 4B,C), whereas only a few
(7%) were found in the CMZ (n=41). By contrast, of the GFP-Meis1
clones located in the central retina (72%, n=39), none contained
Islet1-positive cells at this stage (Fig.
4E). In addition, a large portion of these Meis1-expressing clones
(28%, n=39) was found in the CMZ
(Fig. 4F). The fact that
Meis1-expressing cells were always found in undifferentiated regions of the
neuroepithelium, leads us to conclude that maintenance of meis1
expression in the first 48 hours of eye development is incompatible with
neuronal differentiation.

meis1 is required for the G1-S transition and the expression of
the G1-S regulators cyclin D1 and c-myc. (A-D)
Histograms displaying DNA content (cell cycle profile) of cells from dissected
19hpf eyes of meis1-MO treated embryos relative to (A) controls
(Crtl-MO), (B) meis1-MO+GFP-meis1 mRNA, (C)
meis1-MO+cyclin D1 mRNA, and (D)
meis1-MO+c-myc mRNA injected embryos. Number of replicates
(n) and P values are indicated. meis1 knockdown
induces a delay in G1, which is partially rescued by co-injection of
GFP-meis1 (B), cyclin D1 (C) and c-myc (D) mRNAs.
(E) Average percentages are shown for G1, S and G2/M DNA content. The
cell-cycle profiles of control morphants and of uninjected, wild-type embryos
are indistinguishable (not shown). (F-I) In situ analysis of cyclin
D1 and c-myc transcription in control (F,H) and meis1
morphants (G,I) at 19hpf. meis1 morphants show a dramatic reduction
in cyclin D1 and c-myc in the eye (red arrowheads).
cyclin D1 and c-myc are still detected in other body regions
(black arrowheads). Hybridization and reaction development were performed
strictly in parallel. Representative embryos are shown.

Clonal overerexpression of meis1 in the developing eye prevents
differentiation and results in cell sorting. Single optical sections from
confocal z-stacks of GFP (A-C) or GFP-Meis1 (D-F)
expressing clones induced genetically in developing eyes. GFP-meis1
signal is nuclear. At 24-30hpf, both clone types frequently span the whole
width of the neuroepithelium (A,D). Confocal optical sections through the
central retina (B,E) and z-sections (C,F) of 48hpf eyes. At this
stage, GFP clones comprise both Islet1-expressing and non-expressing cells
(B,C). By contrast, same-stage GFP-Meis1 clones in the central retina do not
contain Islet1-positive cells (E). GFP-Meis1 clones are often located in the
CMZ (F). The arrowheads (C,F) point to the CMZ, and the retina and the lens
(L) are outlined.

Our results indicate that, during early eye development, meis1
shares two roles with its fly homolog, hth. First, meis1 is
required to maintain proliferation of the multipotent cells of the early eye,
by promoting the G1-to-S transition of the cell cycle. Mechanistically,
meis1 regulates the transcription of at least two potent cell-cycle
activators: cyclin D1 and c-myc. Second, ath5
follows receding meis1 expression in a similar fashion as in
Drosophila, where hth expression retracts as the
atonal-expressing differentiation wave advances. This finding is in
accordance with a model in which the expression of meis1 has to be
downregulated to allow further differentiation of the fish retina, and agrees
with our results that the sustained expression of meis1 is
incompatible with neural differentiation. Similar results have been obtained
in chicken and mouse by Heine and co-workers
(Heine et al., 2008). Although
retinal lamination in meis1 morphants is not grossly affected, we
cannot rule out specific effects on the specification and/or differentiation
of specific retinal cell types, as meis1, together with
meis2.1 and meis2.2, is redeployed in postmitotic cells of
the ganglion cell and inner nuclear layers
(Fig. 1M and see Fig. S3 in the
supplementary material).

The expression of meis1 in the CMZ, and the fact that forcing
meis1 expression results in the localization of the expressing cells
to the CMZ, suggest that meis1 might function in specifying the
retinal stem cells of the zebrafish. In this regard, it is interesting to note
that meis1 expression resembles that of Pax6, a previously
described retinal progenitor transcription factor
(Raymond et al., 2006)
(reviewed by Amato et al.,
2004). In Drosophila, previous results showed that
hth and eyeless are co-expressed in the undifferentiated
domain and that their products might directly interact in vivo
(Bessa et al., 2002). All these
results seem to indicate that a common molecular mechanism to maintain a
multipotent stem-like state exists during eye development in vertebrates and
invertebrates.

In addition to controlling several developmental processes, Meis genes are
overexpressed in an increasing number of cancer types
(Lawrence et al., 1999;
Segal et al., 2004;
Geerts et al., 2005;
Dekel et al., 2006). Therefore,
the identification of functional targets of the Meis genes involved in the
maintenance of the undifferentiated and proliferative state during normal
development, such as cyclin D1 and c-myc, is likely to be
instrumental in deciphering the mechanisms underlying Meis-associated
tumors.

Supplementary material

Acknowledgments

We are grateful to Dorothea Schulte for communicating results prior to
publication. This work was supported by grants BMC2003-06248 and
BFU2006-00349/BMC from the Spanish Ministry of Education and Science,
co-funded by FEDER, to F.C. J.B., M.J.T. and J.S. are supported by the
Fundação para Ciência e Tecnologia, Portugal. The CABD is
institutionally supported by Junta de Andalucía.

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